Characterization of Ca2+ influx through recombinant P2X receptor in C6BU-1 cells

1 The effects of exogenous adenosine 5'-triphosphate (ATP) and alpha,beta-methylene ATP (alpha,beta meATP) on C6BU-1 cells transfected with P2X(2) and P2X(3) subtypes, separately or together (P2X(2+3)), were investigated using fura-2 fluorescence recording and whole-cell patch clamp recording methods. 2 Untransfected C6BU-1 cells showed no intracellular Ca2+ ([Ca2+](i)) increase in response to depolarizing stimulation with high K+ or stimulation with ATP. There was no current induced by ATP under voltage clamp conditions in untransfected C6BU-1 cells. ATP caused Ca2+ influx only from extracellular sources in C6BU-1 cells transfected with the P2X subtypes, suggesting that the C6BU-1 cell line is suitable for the characterization of Ca2+ influx through the P2X subtypes. 3 In C6BU-1 cells transfected with the P2X(2) subtype, ATP (more than 10 mu M) but not alpha,beta meATP (up to 100 mu M) evoked a rise in [Ca2+](i). 4 In the cells transfected with the P2X(3) subtype, current responses under voltage clamp conditions were observed at ATP concentrations higher than 0.1 mu M of alpha,beta meATP were required. This discrepancy in the concentration dependence of the agonist responses with respect to the [Ca2+](i) rise and the current response was seen only with the P2X(3) subtype. In addition, the agonist-induced rise in [Ca2+](i) was observed only after the first application because of desensitization of this subtype. 5 In C6BU-1 cells co-transfected with P2X(2) and P2X(3), ATP at 1 mu M evoked a [Ca2+](i) rise. This responsiveness was higher than that of the other subtype combinations tested. The efficiency of expression was improved by co-transfection with P2X(2) and P2X(3), when compared to transfection with the P2X(3) subtype alone. The desensitization of the P2X(2+3) was apparently slower than that of the P2X(3) subtype alone. Therefore, this combination could respond to the repeated application of agonists each time with a [Ca2+](i) rise. 6 These results suggest that the P2X(2) and P2X(3) subtypes assemble a heteromultimer and that this heterogeneous expression acquires more effective Ca2+ dynamics than that by homogenously expressed P2X(2) or P2X(3).